Polynucleotide separation meyhod and apparatus therefor

ABSTRACT

Different probes each having a specific base sequence are immobilized to each of independent areas formed on the surface of a substrate, complementary polynucleotides in a sample solution are hybridized to the probes, and each of the independent areas on the substrate is heated and then cooled in sequence, and hence the solution is recovered to extract different polynucleotides separately corresponding to individual probes.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for selectivelyextracting a target polynucleotide having a specific base sequence froma polynucleotide mixture sample having a plurality of differentsequences or from cells, and an apparatus therefor.

[0002] A DNA chip is one of means for detecting a specific base sequenceof DNA quickly and easily. It utilizes micropatterning techniques usedin microprocessing of semiconductors and the complementarity of DNA. Inthis technique, DNA sequence of a sample solution is analyzed in thefollowing manner: Single stranded-oligonucleotides as probes havingdifferent sequences each of a length of approximately 8-9 bp areimmobilized separately onto each of two-dimensionally split individualareas on a substrate; the sample solution containing DNA is addeddropwise to the substrate to hybridize the DNA separately with each ofprobes in each of the areas of the substrate while attaching afluorescent dye to the hybrids simultaneously in the hybridization step;and the magnitude of the hybridization between the probes and DNA in thesample solution is optically determined through emitted fluorescence toanalyze the DNA sequence in the sample solution.

[0003] U.S. Pat. No. 4,446,237 discloses a method for capturing a targetoligonucleotide (DNA or RNA) as a probe on a solid phase. According tothis method, the oligonucleotide in a sample solution is denatured intosingle strands by heating, which is then immobilized on the surface of anitrocellulose membrane. S. R. Rasmussen et al. describe another methodfor capturing a target oligonucleotide sample on a solid phase inAnalytical Biochemistry 198, 128-142(1991). According to the method, thephosphate group at the 5′-end is activated by using 1-methylimidazoleand 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The activatedpolynucleotide is then immobilized onto a polystyrene microplate havinga secondary amine on its surface. The activated 5′-end phosphate groupreacts with the secondary amine, so that the 5′-end of thepolynucleotide is covalently immobilized onto the microplate surface.

[0004] As thus described, a target polynucleotide in a sample solutioncan be captured and analyzed by selectively hybridizing a targetpolynucleotide (DNA or RNA) with a complementary oligonucleotideimmobilized as a probe on a membrane surface. The DNA chip is on thebasis of this concept.

[0005] Okano et al. describe a method for extracting targetpolynucleotides by hybridizing the target polynucleotides to probesimmobilized on a chip, heating the chip to denature the capturedpolynucleotides on the probes to separate and collect them from the chipin U.S. Pat. No. 5,607,646.

[0006] A method for selectively extracting a target polynucleotide byutilizing difference in rates in electrophoresis of polynucleotides ingel has been in wide use.

SUMMARY OF THE INVENTION

[0007] The gene analysis technology using a DNA chip described in theabove is a technique for hybridizing and analyzing a targetpolynucleotide (DNA or RNA) by complementarily hybridizingsingle-stranded polynucleotides derived from the target polynucleotidein a sample solution with a probe (a specific singlestranded-oligonucleotide) with a length of 8-9 bp formed on a substrate.This technique is never directed to further extract the captured orhybridized DNA or RNA single stranded-polynucleotide on the probe fromthe substrate selectively.

[0008] Conventional techniques give no consideration of extracting atarget polynucleotide to be extracted directly from cells.

[0009] According to conventional separating techniques usingelectrophoresis, the mobility of each of polynucleotides is relative toeach other and fluctuates with changes of electrophoresis conditions,and thus identification of an extracted sample solution component isrequired. In addition, exact separation and purification of a tracequantity of a target polynucleotide is difficult because diffusion inthe electrophoresis step can invite contamination of polynucleotideswith each other.

[0010] It is, therefore, an object of the present invention to provide aprocess and apparatus for selectively extracting a trace quantity of atarget polynucleotide (DNA or RNA) having a specific base sequencerapidly with a high precision.

[0011] The invention proposes, to achieve the above object, selectiveextraction of a target polynucleotide alone from a sample solution bymodifying each of independent split areas on the surface of a substrateseparately with each of probes (specific oligonucleotides) havingdifferent base sequences respectively, hybridizing polynucleotides (DNAor RNA) in the sample solution separately to the probes, and thenheating a specific area alone of the substrate selectively to allow apolynucleotide alone complementarily hybridized with the heated probe toliberate from the probe.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features, objects, and advantages of the presentinvention will become apparent upon a consideration of the followingdescription of the invention when read in conjunction with the drawings,in which:

[0013]FIG. 1 is a gene processing flowchart for demonstrating where themethod and apparatus for separating polynucleotides according to theinvention are located in analysis and selective extraction process ofgene;

[0014]FIG. 2 is a schematic diagram illustrating a basic configurationaccording to a first embodiment of the invention;

[0015]FIG. 3 is a schematic diagram illustrating a first means forheating a specific area on a substrate of the first embodiment;

[0016]FIG. 4 is a schematic diagram illustrating a second means forheating a specific area on the substrate according to the firstembodiment;

[0017]FIG. 5 is a schematic diagram illustrating a third means forheating a specific area on the substrate according to the firstembodiment;

[0018]FIG. 6 is a schematic diagram illustrating a practicalconfiguration of a polynucleotide separation cell according to the firstembodiment;

[0019]FIG. 7 is a sectional view along with the lines A-A of the cellillustrated in FIG. 6;

[0020]FIG. 8 is a sectional view along with the lines B-B of the cellillustrated in FIG. 6;

[0021]FIGS. 9A, 9B and 9C are timetables respectively illustrating thenucleotide separation process according to the first embodiment of theinvention;

[0022]FIG. 10 is a diagram illustrating the relationship between asubstrate 1 having a target polynucleotide hybridization area and anoptical system for detecting the hybridization in the targetpolynucleotide hybridization area, according to a second embodiment ofthe invention;

[0023]FIG. 11 is a sectional view along with the lines A-A of thesubstrate 1 shown in FIG. 10;

[0024]FIGS. 12A, 12B and 12C are sectional views along with the linesB-B′, C-C and D-D, respectively, of a temperature control unit 133 ofthe substrate 1 according to the second embodiment.

[0025]FIG. 13 is a block diagram illustrating a polynucleotideseparation cell 251 and related devices thereto according to the secondembodiment;

[0026]FIG. 14 is a sectional view along with the lines G-G of thepolynucleotide separation cell 251 according to the second embodiment;

[0027]FIG. 15 is a timetable demonstrating the separation process in thepolynucleotide separation cell according to the second embodiment;

[0028]FIG. 16 is a diagram illustrating stable immobilization of probeson a substrate 1 according to a third embodiment of the invention;

[0029]FIGS. 17A, 17B and 17C are diagrams respectively illustrating testresults for verifying the advantages of the substrate according to thethird embodiment;

[0030]FIGS. 18A and 18B are diagrams illustrating efficient extractionof a target oligonucleotide hybridized to a probe on the substrateaccording to the third embodiment;

[0031]FIG. 19 is a diagram schematically illustrating results ofelectrophoresis, demonstrating the fractionation of a doublestranded-polynucleotide according to the third embodiment;

[0032]FIG. 20 is an external view of a capillary applicable to a fourthembodiment of the invention;

[0033]FIG. 21 is a sectional view along with the lines A-A of thecapillary applicable to the fourth embodiment;

[0034]FIG. 22 is a diagram of an illustrative configuration forimmobilizing a probe to a target polynucleotide hybridization area;

[0035]FIG. 23 is a general view of the configuration of a polynucleotideseparation apparatus according to the fourth embodiment;

[0036]FIG. 24 is a sectional view along with the lines B-B of apolynucleotide separation module 431 of the fourth embodiment;

[0037]FIG. 25 is a diagram illustrating a variation of thepolynucleotide separation module 431 according to the fourth embodimentshown in FIG. 24;

[0038]FIG. 26 is a diagram illustrating another variation of thepolynucleotide separation module 431 according to the embodiment shownin FIG. 24;

[0039]FIG. 27 is a diagram illustrating a variation of thepolynucleotide separation module 431 according to the embodiment shownin FIG. 26;

[0040]FIG. 28 is a diagram illustrating basic elements of a fifthembodiment of the invention;

[0041]FIG. 29 is a diagram illustrating a basic configuration of thefifth embodiment including a combination of a polynucleotide separationcell having the basic elements illustrated in FIG. 28 and an opticalsystem;

[0042]FIG. 30 is a sectional view along with the lines A-A of theseparation cell illustrated in FIG. 29;

[0043]FIG. 31A is a diagram illustrating the state where targetpolynucleotide hybridization areas are formed on the surface of thesubstrate illustrated in FIG. 28;

[0044]FIG. 31B is a diagram illustrating the state where a blood sampleis introduced into the polynucleotide separation cell and white bloodcells float over the surface of the substrate;

[0045]FIG. 31C is a diagram illustrating the state where white bloodcells are attracted to the target polynucleotide hybridization areas byan alternating field and placed separately on each of areas;

[0046]FIG. 31D is a diagram illustrating the state where white bloodcells capable of making antibody response to antigen substances aloneare labeled and become to emit fluorescence;

[0047]FIG. 31E is a diagram illustrating the state where only one whiteblood cell of those hybridized on the individual target polynucleotidehybridization areas is destroyed and disappears from the substrate;

[0048]FIG. 32A is a diagram illustrating the state where probes areimmobilized on one target polynucleotide hybridization area;

[0049]FIG. 32B is a diagram illustrating the state where a white bloodcell is inducted and hybridized to the target polynucleotidehybridization area;

[0050]FIG. 32C is a diagram schematically illustrating the state wherepolynucleotides and proteins migrate by electrophoresis respectively inthe opposite direction to each other; and

[0051]FIG. 32D is a diagram schematically illustrating the state wherepolynucleotide not hybridized to probes of the target polynucleotidehybridization area migrate by electrophoresis.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0052]FIG. 1 is a gene processing flowchart for demonstrating where themethod and apparatus for separating polynucleotides according to theinvention are located in analysis and selective extraction process ofgene. Processes 801 and 802 are preparative processes respectively forsample cell introduction and sample solution introduction, respectively.The present invention can be applied both to cells and solutions assamples. Process 803 is a cell component extraction process. Process 804is a PCR amplification process, being conducted when a targetpolynucleotide has a low concentration. Process 805 is a process forsample DNA selection on a plurality of independent areas, whichcharacterizes the invention. Process 806 is a selected sampleobservation analysis process. Process 807 is sample DNA extractionprocess from the independent areas.

[0053] According to the invention which includes a process for sampleDNA selection on a plurality of independent areas and wherein each DNAis extracted from each of the areas, efficient fractionation can beachieved, as described in detail hereinbelow.

[0054] Embodiment I

[0055]FIG. 2 is a schematic diagram illustrating a basic configurationof a first embodiment of the invention. Substrate 1 is for hybridizingand selecting a target polynucleotide to be extracted on its surface,comprising substrate base 13 and target polynucleotide hybridizationareas 14 which are two-dimensionally mounted on the surface of thesubstrate base 13 by splitting to the order of 1 micrometer square bymeans of a light exposing technique. A plurality of probes (singlestranded-oligonucleotides) having a different sequence for each areawith a length of about 8-9 bp are individually immobilized on thesurface of each of the target polynucleotide hybridization areas 14. Theprobes are oligonucleotides complementary to a target polynucleotide ina sample solution. When the sample solution reaches the surfaces of thetarget polynucleotide hybridization areas 14, the probes hybridize withthe target polynucleotide. In other words, the target polynucleotide iscaptured by the probes. Upon the hybridization between the probes andthe target polynucleotide, a fluorescent dye simultaneously attaches tothe hybrids. Heater 31 is provided on the back of the substrate 1 andcapable of heating the whole surface of the substrate 1 from around roomtemperature up to about 95° C. The substrate 1 is disposed in thedirection perpendicular to the optical axis of object lens 15. Each oflight source 16 for fluorescence observation and infrared laser source17 for convergent light beam irradiation is disposed in the directionperpendicular to the optical axis of the object lens 15. Dichroicmirrors 181, 182 are disposed at the positions where the optical axis ofthe object lens 15 intercepts with the optical axes of the light sources16 and 17 respectively, to induce lights irradiated from the lightsources 16 and 17 respectively to the objective lens 15. The light fromthe light source 16, the fluorescence from the florescent dye and thelight from the light source 17 may preferably have a differentwavelength from each other. The light from the light source 16 can beconverged through bandpass filter 151 to adequate wavelengths forexciting the fluorescent dye. The exciting light induced to theobjective lens 15 is converged to excite the fluorescent dye attached tothe target polynucleotide hybridized and selected on the substrate. Theexcited fluorescent dye emits fluorescence in an intensity proportionalto each amount of the target polynucleotide hybridized to probes of eachof the individual areas of the substrate. Mirror 183 is to reflect alight beam which is obtained by collecting the emitted fluorescencethrough the objective lens 15. Emission filter 152 is to selectivelytransmit only a light beam having a wavelength range of fluorescence tobe detected. Detector 191 is to detect the intensity of the fluorescencetransmitted through the emission filter 152.

[0056] The spatial distribution of the amount of the targetpolynucleotide in the sample solution which has been hybridized to theprobe immobilized on the substrate can be obtained as a fluorescenceimage by moving mutually the objective lens 15 and the substrate 1 andscanning the surface of the substrate 1 in sequence with the excitinglight induced through the objective lens 15 to find the spatialdistribution of the fluorescence emission intensity emitted by thefluorescent dye. When the fluorescent dye attached with the targetpolynucleotide is excited for observation, the use of an exciting lighthaving a wavelength in the visible region and a fluorescent dye capableof fluorescing by means of a visible light are preferred in theobservation of the fluorescence from the dye attached with the targetpolynucleotide. An autoemission fluorescence of the targetpolynucleotide can also be observed instead of light emission of thefluorescent dye attached to the target polynucleotide. In this case theexciting light preferably has a wavelength of near UV region rangingfrom 280 nm to 400 nm.

[0057] The fluorescence emission intensity of the fluorescent dyedecreases in proportion to an increasing temperature through thefunction of the thermal quenching effect. By using this phenomena, anarea specific temperature increase in the target polynucleotidehybridization areas on the substrate can be determined according to thepresent embodiment. According to the apparatus illustrated in FIG. 2,changes of the fluorescence emission intensity of the fluorescent dyeattached with the target polynucleotide, which has been detected by thedetector 191 can be analyzed with analyzer 192 based upon thetemperature-dependency of changes of the fluorescence emissionintensity, and the temperature of the target polynucleotidehybridization areas can thus be estimated.

[0058] An infrared light emitted by the infrared laser source 17 isreflected on the dichroic mirror 182 downward, converged on theobjective lens 15 and irradiated to a target polynucleotidehybridization area 14 formed on the substrate 1. In the targetpolynucleotide hybridization area 14, a thin film layer or particlelayer formed on the substrate 1 absorbs infrared light to evolve heatand thereby to area-specifically increase the temperature in the samplesolution. The convergent position of the light from the laser source 17converged on the objective lens 15 on the surface of the substrate 1 canbe changed arbitrarily by moving the objective lens 15 or moving thesubstrate 1. To avoid damage to the hybridized target polynucleotide,the use of a light having a wavelength region not to be absorbed by thetarget polynucleotides is preferred. By way of illustration, nearinfrared light having a wavelength of equal to or more than 800 nm orvisible light having a wavelength of equal to or more than 400 nm ispreferably used.

[0059] It is known that a single stranded-polynucleotide such as DNA orRNA, being complementary to another single stranded-polynucleotide suchas a probe, forms base pairs through hydrogen bonds with thecomplementary polynucleotide to form a double stranded-polynucleotide;and that the double stranded-polynucleotide formed by base pairs throughhydrogen bonds denatures to form single stranded-polynucleotides whenthe temperature of a sample solution is increased up to about 95° C.Accordingly, by irradiating the convergent infrared ray to a specifictarget polynucleotide hybridization area 14 on the substrate to increaseits temperature up to 95° C., the target polynucleotide hybridized tothe probe on the specific target polynucleotide hybridization area canselectively separated.

[0060] The probes on the substrate 1 and polynucleotides in the samplesolution above the substrate 1 can be elongated when a planer electrodeis placed on the substrate 1 and an alternating field is applied betweenthe planer electrode and the substrate 1. Consequently, nonspecifichybridization between the probes and polynucleotides in the samplesolution can be reduced and denaturation of the pairs between the probesand the target polynucleotide through the convergent light canefficiently be achieved.

[0061] The denaturing temperature of the target polynucleotideshybridized to the probes increases in proportion to the amount ofhydrogen bonds therebetween, and polynucleotides can selectively befractionated in accordance with the magnitude of hybridization matchingbetween the target polynucleotides and the probes by controlling thedenaturing temperature. In other words, the target polynucleotideshybridized to the probes begin to denature with an gradually increasingtemperature, whereas the less the polynucleotides have hydrogen bonds,the earlier they begin to denature. The target polynucleotides can,therefore, be selectively fractionated in consideration with themagnitude of specific bonds by feedback-controlling the output of thelaser source 17 based upon the temperature information obtained throughthe analyzer 192 and adjusting the temperature of the targetpolynucleotide hybridization areas 14.

[0062] The variations of the denaturing temperatures of targetpolynucleotides hybridized to probes can be minimized by adding atertiary ammonium salt such as tetramethylammonium chloride to thesample solution.

[0063]FIG. 3 illustrates a first means for heating a specific area ofthe target polynucleotide hybridization areas 14 on the substrate.According to the present embodiment, substrate base 13 is composed ofelectrically conductive film 131 provided on the surface side andthermally conductive insulating substrate 132. Onto the electricallyconductive film 131 are placed a plurality of target polynucleotidehybridization areas 141, 142, 143, 144, 145 and 146. Each of the targetpolynucleotide hybridization areas has a dual-layer structure composedof photoabsorbing layer 21 such as an aluminium oxide thin film andheat-insulating layer 22. Probes (oligonucleotides) 41, 42, 43, 44, 45and 46 each having a length of 8-9 bp are respectively immobilized tothe surf ace of each of the target polynucleotide hybridization areasseparately. FIG. 3 schematically illustrates the state where targetpolynucleotides 41′, 42′, 43′ and 46′ are respectively hybridized onlyto the probes 41, 42, 43 and 46 of these probes. By identifying thetarget polynucleotide hybridization areas through fluorescenceobservation, the complementarity of the target polynucleotideshybridized to the probes among polynucleotides in the sample solutioncan be estimated. In the embodiment illustrated in the figure, theprobes in the areas 141, 142, 143 and 146 complementarily hybridize tothe target polynucleotides, whereas those in the areas 144 and 145 donot, indicating that there is no polynucleotide having complementaryrelation with the probes in the areas 144 and 145.

[0064] Divergent light 51 is then irradiated through the objective lens15 to the target polynucleotide hybridization area 142 where the targetpolynucleotide 42′ is hybridized to the probe 42; the photoabsorbinglayer 21 in the area 142 absorbs the convergent light 51 and evolvesheat. The heat from the photoabsorbing layer 21 in the area 142 allowsthe vicinity of the area 142 to increase its temperature up to about 95°C., and hence hydrogen bonds between the probe 42 and the targetpolynucleotide 42′ are dissociated to denature the target polynucleotide42′ alone which has been hybridized to the area 142. When the size of anarea where the convergent light is converged is smaller than that of aunit target polynucleotide hybridization area, the light axis should beadjusted to ensure that the convergent area is within the targetpolynucleotide hybridization area. When a unit target polynucleotidehybridization area has a smaller size than the divergent area of thedivergent light, individual areas should preferably be arranged in sucha manner that gaps between individual target polynucleotidehybridization areas are sufficient and only one area is to be heated bythe convergent light. In FIG. 3, only one probe is shown in each targetpolynucleotide hybridization area to be easy to read, but in practice, aplurality of probes having an identical base sequence are generallyimmobilized to each area.

[0065] According to the present embodiment, where the electricallyconductive film 131 is placed on the surface of the substrate base 13,probes and/or polynucleotides can be elongated by providing a counterelectrode plate (not shown) above the substrate 1 and applying analternating field between the electrode plate and the substrate, asdescribed above. By this configuration, erroneous hybridization in thehybridization step and steric entanglement in the denaturation of thetarget polynucleotide from the probe by heating the targetpolynucleotide hybridization area can be prevented to ensure anefficient processing.

[0066] In addition, the cooling effect through the absence of theconvergent light 51 irradiation can be improved by using the thermallyconductive insulating substrate 132 as the substrate base 13.

[0067] The photoabsorbing layer 21 according to the present embodimentmay be prepared by vapor deposition, as well as by coating or spraying.The target polynucleotide hybridization areas can be formed in the shapeof square or rectangular, as well as round or ellipse in accordance withthe shape of the convergent light.

[0068]FIG. 4 illustrates a second means for heating a specific area onthe substrate 1. The photoabsorvable thin layer 21 is formed on thetarget polynucleotide hybridization areas in the embodiment of FIG. 3,whereas, in the present embodiment, particles 23 each havingphotoabsorbing characteristics and have sufficiently small sizes incomparison with those of the target polynucleotide hybridization areasare dispersed and placed on the target polynucleotide hybridizationareas. At least one particle should be placed on each area. According tothe present embodiment, heat insulating layer 22 is separately providedin each of individual areas and the particles 23 are placed onto theupper surface of the insulating layer 22. The substrate 1 comprisessubstrate base 13 composed of electrically conductive film 131 andthermally conductive insulating substrate 132 as well as in theembodiment illustrated in FIG. 3.

[0069] When the convergent light 51 is irradiated to a specific targetpolynucleotide hybridization area 142, the particle 23 in the areaabsorbs the light to evolve heat and hence the vicinity of the area 142alone is increased in temperature so that the target polynucleotidealone being hybridized to the probe on the area 142 can be denaturedfrom the probe. According to the present embodiment, the use ofparticles 23 ensures an area, smaller than the convergent range of theconvergent light of the present embodiment of FIG. 3, to be heatedspecifically, whereas the size of each target polynucleotidehybridization area and that of the convergent area of the convergentlight have a similar relation as in the embodiment of FIG. 3. Thephotoabsorbing layer composed of the particles 23 according to thepresent embodiment can be prepared by coating or spraying of particles.The particles can be shaped arbitrarily as in the embodiment of FIG. 3.

[0070]FIG. 5 illustrates a third means for heating a specific area onthe substrate 1. The present embodiment is similar to the embodiment ofFIG. 4 in that temperature increase by particle 24, which absorbs lightat wavelength region of the convergent light, is utilized, but differentfrom the latter in that the particle 24 is captured by the convergentlight 51 in the manner of optical forceps, and the captured particle 24is placed in a target polynucleotide hybridization area on which probeand the target polynucleotide are to be denatured. The numericalaperture of the objective lens 15 in this case should preferably beequal to or more than 1.2 for ensuring satisfactory characteristics asoptical forceps. Heat insulating layer 22 is also provided respectivelyin each area in the present embodiment. Substrate 1 comprises substratebase 13 composed of electrically conductive film 131 and thermallyconductive insulating substrate 132 as well as in the embodiment of FIG.3. As the embodiment of FIG. 5 is similar to the embodiment of FIG. 4except that the particle 24 is captured in the manner of opticalforceps, other descriptions concerning the figure are omitted.

[0071] According to the present embodiment, temperature increase by theparticle 24 is confined within the vicinity of the particle 24 in thearea where the particle 24 is placed, the sizes of the targetpolynucleotide hybridization areas and that of the convergent area ofthe convergent light have no fundamental relationship. To be morespecific, temperature increase by the particle 24 is limited to thevicinity of the particle 24 in the area where the particle 24 is placed,and hence a target polynucleotide to be denatured is limited to thatpresent in the target polynucleotide hybridization area where theparticle 24 is place, even when the convergent area of the convergentlight covers a plurality of target polynucleotide hybridization areas.The present embodiment is, therefore, advantageous in that the targetpolynucleotide hybridization areas can be arranged with an increaseddensity.

[0072]FIG. 6 demonstrates a more detailed illustrative configurationincluding the relationship between the nucleotide separation cell 7 andthe substrate 1 using the basic configuration of the embodiment shown inFIG. 2.

[0073] Part of the polynucleotide separation cell 7 in FIG. 6 is notchedfor viewing the inside thereof. FIGS. 7 and 8 are sectional views of thepolynucleotide separation cell 7 respectively along with the lines A-Aand the lines B-B.

[0074] The polynucleotide separation cell 7 is provided with upper cellplate 721, lower cell plate 722, and three sample solution chambers 731,732 and 733 which are partitioned by a plurality of spacers 723. Theupper cell plate 721 is fabricated of a light transmittable material.The tree sample solution chambers can import and export a samplesolution through sample solution inlets 711, 712 and 713 respectively.Between each of the sample solution chambers are connected communicationholes 714 and 715, through which the sample solution is transferred. Thesubstrate 1 on which probes are immobilized as illustrated in FIG. 2 ismounted on the lower cell plate 722 in the sample solution chamber 732,and on the substrate 1 is integrated electrode plate 131 for applying anelectric field. Electrodes 751, 752, 754 and 755 are attached toindividual side walls of the sample solution chambers 731, 732 and 733respectively, and mesh electrode 753 is placed on the inner wall of theupper cell plate for applying an electric field onto above the samplesolution chamber 732. The temperature of the substrate 1 can becontrolled within the range from 0° C. to 95° C. through Peltier devices32, 34 and 35 each provided with a cooling plate 33, and individualtemperatures of the sample solution chambers 731, 732 and 733 can beindependently controlled within the range from 0° C. to 95° C. byoperating the Peltier devices 32, 34 and 35 independently. Accordingly,the PCR reaction or denaturation reaction can be carried out in each ofthe sample solution chambers without changing the temperatures of theother chambers. To move the position of the substrate 1 to be observedthrough the objective lens 15, the cell 7 is fixed on rails 742, 743 and744 by jig 741 and can be arbitrarily transferred on a two-dimensionalplane by a stepping motor (not shown).

[0075]FIGS. 9A through 9C illustrate timetables demonstrating apolynucleotide separation process using the polynucleotide separationcell shown in FIGS. 6-8. Initially, a sample solution containingpolynucleotides introduced into the sample solution chamber 731 issubjected to PCR amplification as a pretreatment by applying a total ofabout 20 to 30 cycles of chronological changes in temperature to thesolution as shown in FIG. 9A. In other words, doublestranded-polynucleotides are denatured into singlestranded-polynucleotides in the first denaturation process. Annealingwith probes in the sample solution and subsequently polymeraseelongation are then conducted, and these processes are repeated toamplify polynucleotides. The temperatures in the processes arecontrolled on the whole sample solution in the sample solution chamber731 by the Peltier device 32. From the sample solution containing theamplified polynucleotides, a polynucleotide having a specific basesequence is extracted by means of process as shown in FIG. 9B. To bemore specific, in the sample introduction process, polynucleotides inthe sample solution in the sample solution chamber 731 are induced tothe sample solution chamber 732 by setting the electrodes 751 and 755 ascathodes and the electrode 754 and the electrode above the substrate 1as anodes. The sample solution in the sample solution chamber 732 isthen heated to 95° C. by the Peltier device 34 to disassociate hydrogenbonds in polynucleotides in the sample solution to form singlestranded-polynucleotides. The single stranded-polynucleotides are thenhybridized to probes in the target polynucleotide hybridization area onthe substrate 1 by cooling the solution to a temperature of 37° C.Remained polynucleotides in the sample solution not hybridized to theprobes in the hybridization areas are then returned to the samplesolution chamber 731 by setting the electrode 751 as an anode. The cellis then subjected to fluorescence observation to identify to which areaon the substrate 1 the target oligonucleotide is hybridized. On thebasis of information of an area where the target polynucleotide ishybridized to the probes on the substrate 1 (output of the fluorescentemission intensity detector 191 shown in FIG. 2), the targetpolynucleotide hybridized to the probe in a target polynucleotidehybridization area on the substrate 1 can be dissociated from the probeby irradiating a convergent light to the area. In this process, analternating field is applied between the electrode 753 and the electrode131 on the surface of the substrate 1 to elongate the targetpolynucleotide. In addition, the target polynucleotide in the specifictarget polynucleotide hybridization area on the substrate 1 alone isdenatured by supplying the convergent light 51 from the lens 15, whilesetting the electrode 755 as an anode, and thus the objective targetpolynucleotide alone is introduced into the sample solution chamber 733.By setting the electrode 752 as a cathode while retaining the electrode751 as an anode, polynucleotides in the sample solution in the samplesolution chamber 731 which have not been hybridized to the probes areretained in the sample solution chamber 731 without transferring to thesample solution chamber 732 or 733. Components required for PCR are thenintroduced through the solution inlet 713 to the sample solution chamber733 while continuously applying an electric field to the electrodes.About 20 to 30 cycles of aftertreatment shown in FIG. 9C are thencarried out to extract and amplify an objective polynucleotideselectively with a high precision and a high speed from the samplesolution introduced into the sample solution chamber 733. Afterextracting the amplified sample solution in the sample solution chamber733, each of polynucleotides hybridized to each of the areas can beselectively extracted and amplified in sequence by moving the substrate1 and repeating the separation process shown in FIG. 9B and theaftertreatment PCR process shown in FIG. 9C in a similar manner asabove.

[0076] Specific procedures for decreasing the temperatures are notdescribed in the timetables shown in FIGS. 9A through 9C, whereascooling of individual chambers can be conducted in a short time bypositively using the Peltier devices 32, 34 and 35 in FIG. 6 for heatdissipation. Such Peltier devices can be provided separately inindividual target polynucleotide hybridization areas for cooling afterthe area specific denaturation for denaturing polynucleotides hybridizedto the probes of the target polynucleotide hybridization area.

[0077] Embodiment II

[0078] Embodiment II is essentially identical with Embodiment I exceptthat the former utilizes heating elements embedded in the substrate 1for temperature control of individual target polynucleotidehybridization areas to denature the target polynucleotide hybridized tothe probes, instead of the convergent light irradiation in the latter.The present embodiment will now be described in detail with reference toFIGS. 10 through 15.

[0079]FIG. 10 illustrates the relationship between substrate 1 having atarget polynucleotide hybridization area and an optical system fordetecting hybridization in the target polynucleotide hybridization area,according to the second embodiment of the invention. Comparison betweenFIG. 10 and FIGS. 2 and 3 demonstrates that the substrate according tothe present embodiment comprises substrate base 13 composed ofelectrically conductive film 131 having target polynucleotidehybridization areas on its surface, temperature control unit 133 andthermally conductive insulting substrate 132. On the surface of theelectrically conductive film 131 are provided a plurality of targetpolynucleotide hybridization areas 141, 142, 143, 144, 145 and 146, andinside of the temperature control unit 133 are embedded electrodes andheating elements to evolve heat by the electrodes, for independentlycontrolling the temperatures of individual target polynucleotidehybridization areas separately. Each of the target polynucleotidehybridization areas has a dual-layer structure composed of probehybridization layer 221 such as an aluminum oxide thin film layer, andelectric insulator layer 222. Probes (oligonucleotides) 41, 42, 43, 44,45 and 46 each having a length of 8-9 bp are respectively immobilized tothe surface of each of the target polynucleotide hybridization areas.FIG. 10 schematically illustrates the state where complementary targetpolynucleotides 41′, 42′, 43′ and 46′ alone are respectively hybridizedonly to the probes 41, 42, 43 and 46 of these probes.

[0080] When the present embodiment is configured such that a fluorescentdye is attached simultaneously at the time when the targetpolynucleotides 41′, 42′, 43′ and 46′ are hybridized to probes 41, 42,43 and 46, the magnitude of hybridization between the probe and targetpolynucleotide in each area can be estimated through fluorescenceemission intensity by exciting the fluorescent dye and detectingfluorescence emitted from the dye. In addition, area specifictemperature increase in the target polynucleotide hybridization areascan be determined by using the phenomena where the fluorescence emissionintensity of a fluorescent dye decreases in proportion to an increasingtemperature through the function of the thermal quenching effect. Thetemperatures of target polynucleotide hybridization areas can beestimated by analyzing the fluorescence emission intensity changes ofthe fluorescent dye based upon its temperature dependency. The identicalor equivalent elements to those of EMBODIMENT I are indicated with thesame reference numerals in the present embodiment.

[0081]FIG. 11 is a sectional view of the substrate 1 shown in FIG. 10along with the lines A-A. Substrate 1 has a laminate structure composedof thermally conductive insulating substrate 132, temperature controlunit 133 and electrically conductive film 131. On the surface of the toplayer, electrically conductive film 131, is formed a dual-layerstructure of probe hybridization layer 221 to be target polynucleotidehybridization area, and electrically insulating layer 222. Below thetemperature control unit 133 are formed layers of planar electrodes 226,heating elements 225 and planar electrodes 224. Individual electrodes inthe planar electrode 226 and the planar electrode 224 are in the shapeof cross-matrix such that individual crossing points separatelycorrespond to the position of individual heating elements and topositions of individual target polynucleotide hybridization areas.Thermistors 231 for temperature measurement are embedded in thethermally conductive insulating substrate 132 corresponding to each ofthe target polynucleotide hybridization areas, by which the temperatureof each of the hybridization areas can be determined.

[0082]FIGS. 12A through 12C are sectional views of the temperaturecontrol unit 133 of the substrate 1 according to the second embodimentalong with the lines B-B, lines C-C and lines D-D, respectively. Theplanar electrodes 226 are orthogonal to the planar electrodes 224, andindividual crossing points therebetween sandwich each of the heatingelement layers 225. Consequently, a heating element layer 225 alone at acrossing point of a planar electrode 226 and a planar electrode 224 canevolve heat by selectively applying a potential difference between theabove planar electrode 226 and the above planer electrode 224 to pass anelectric current. In other words, by selecting a planar electrode 226and a planar electrode 224 properly and applying a potential differencetherebetween, the temperature of a specific target polynucleotidehybridization area corresponding to the crossing point between theseselected electrodes can be increased.

[0083] The substrate 1 shown in FIG. 10 can be used in place of thesubstrate 1 of the polynucleotide separation cell 7 shown in FIGS. 6through 8, whereas a simpler embodiment will be described herein.

[0084]FIG. 13 is a block diagram illustrating a polynucleotideseparation cell 251 and related devices thereto, and FIG. 14 is asectional view of the polynucleotide separation cell 251 along with thelines G-G, each according to the second embodiment. The polynucleotideseparation cell 251 is composed of a light-transmittable case to permitoptical observation of the substrate 1 in the cell. To the cell 251 areconnected sample solution injection tube 252 for injecting a samplesolution containing polynucleotides, and extraction tube 253 forextracting the target polynucleotide hybridized to a specific targetpolynucleotide hybridization area on the substrate 1, through which thesolution can be passed in the direction indicated by arrows 57 and 58.Above the cell 251 is provided transparent electrode (not shown) forapplying a potential difference between the transparent electrode andthe electrically conductive film 131 of the substrate 1. The presentembodiment includes direct current power source 254, alternating currentpower source 255 and control circuit 256 for allowing a heating elementcorresponding to a specific target polynucleotide hybridization area toevolve heat or for generating a DC field or alternating field in thetarget polynucleotide hybridization area. The state of the substrate 1can, therefore, be controlled on the basis of temperature informationfrom the thermistors 231 according to procedures mentioned below.

[0085]FIG. 15 is a timetable illustrating a separation process in thepolynucleotide separation cell according to the second embodiment.

[0086] From a sample introduced into the cell 251, a polynucleotidehaving a specific base sequence is singly extracted in the process shownin FIG. 15.

[0087] (1) In the sample solution introduction process, polynucleotidesin the solution are induced to the surface of the substrate 1 by settingthe electrically conductive film 131 of the substrate 1 as an anode andthe transparent electrode on the upper surface of the cell as a cathode.Separately, polynucleotides can be elongated by generating analternating field between the electrically conductive film 131 of thesubstrate 1 and the transparent electrode on the upper surface of thecell. In this step, the surface temperature of the substrate 1 isincreased up to 95° C. by allowing all electrodes 224 and 226 to beconnected and to thereby all heating elements 225 to evolve heat, eachin the temperature control unit 133.

[0088] (2) The temperature is then decreased to 37° C. This procedureallows the probes immobilized on the target polynucleotide hybridizationareas to hybridize to the target polynucleotides in the sample solution.

[0089] (3) Next, the surface layer of the substrate 1 is heated up toabout 72° C., and the electrically conductive film 131 of the substrate1 and the transparent electrode on the upper surface of the cell arerespectively set to a cathode and an anode so as to remove, togetherwith the sample solution, polynucleotides non-specifically hybridized toprobes.

[0090] (4) After replacing the solution in the cell 251 with a new one,each one of the electrodes 224 and 226 is respectively allowed to beconnected, and one of the heating elements 225 is allowed to evolveheat, each in the temperature control unit 133 to increase thetemperature of one of the target polynucleotide hybridization areas upto 95° C., thereby the target polynucleotide hybridized to probes inthis area is denatured and extracted with the solution.

[0091] (5) By repeating similar procedures on each of the targetpolynucleotide hybridization areas, each of target polynucleotideshybridized to probes in individual areas can be extracted in sequence.

[0092] In this case, the processing efficiency can be enhanced bydetecting, beforehand, hybridization state of target polynucleotides onindividual target polynucleotide hybridization areas, on the basis oftemperature information from the detector 191 and the analyzer 192 andconducting the temperature control only on an area to which apolynucleotide to be extracted is hybridized, as described in EMBODIMENTI.

[0093] Embodiment III

[0094] EMBODIMENT III relates to a configuration of substrate 1 havingtarget polynucleotide hybridization areas which are designed toimmobilize probes with stability. To be more specific, the presentembodiment proposes the substrate 1 having a configuration in which thesurfaces of hybridization areas are fabricated of oxidized metal filmsand the probes can be immobilized with stability by a silane couplingreaction.

[0095] The substrate 1 according to the present embodiment may beprepared in the following manner: A 0.4-mm thick and 24-mm square glasssubstrate is immersed in an NaOH (1 M) solution and subjected toultrasonic cleaning for 30 minutes. The cleaned substrate is washed withrunning ultrapure water and thereafter baked at 110° C. for 15 minutes.Using a vacuum metallizer, chromium (Cr) is vacuum-deposited on thesubstrate in a thickness of 3 nm, and the deposited substrate is washedwith ethanol. After immersed in 3-glycidoxypropylmethoxysilane (notdiluted) for 5 minutes, the substrate is immersed in a 4%3-glycidoxypropylmethoxysilane solution in 50% ethanol medium for 30minutes with stirring at times. The substrate is then taken out from thesolution and baked at 110° C. for 30 minutes to introduce glycidoxygroups onto the surface of the metal through the silane couplingreagent, and thereby substrate 1 is obtained.

[0096]FIG. 16 illustrates the stable immobilization of probes on asubstrate 1 according to EMBODIMENT III. The substrate 1 according tothe present embodiment is composed of glass substrate 302, Cr layers 303deposited on the substrate 302, which surfaces constitute oxidized films304. Probes are immobilized through silane coupling layers 305. Toverify that the probes are immobilized with stability according to thepresent embodiment, polynucleotides respectively having lengths of 521bp and 625 bp, whose ends are labeled with fluorescent dyesulforhodamine 101, are hybridized to probes 311 and 312, which probesare respectively immobilized through silane coupling layer 305 on targetpolynucleotide hybridization areas 306 and 307.

[0097] A comparative test to verify the advantages of the substrate 1according to EMBODIMENT III will be described below.

[0098] Initially, comparison was conducted among the substrate accordingto EMBODIMENT III, a substrate obtained by vacuum-depositing aluminium(Al) instead of Cr on the surface of glass 302, and a substrate obtainedby subjecting the surface of glass 302 directly to silane coupling. Asthe substrate according to the present embodiment, two substrates wereprepared by vacuum-depositing Cr on the whole surface of glass 302 in athickness of 5.4 nm. These substrates had light transmittance at 600 nmto 800 nm of 53% to 56%. The Al-deposited substrate was obtained byvacuum-depositing Al instead of Cr on the whole surface of glass 302 ina thickness of 15 nm in a similar manner to that in the embodiment. Thesubstrate had a light transmittance at 600 nm to 800 nm of 19 to 25%.Glass itself without deposition of a metal film has a lighttransmittance at 600 nm to 800 nm of about 84%. Within these ranges oftransmittance as above, a target polynucleotide hybridized to a probecan be detected through fluorescence emitted by exciting the targetpolynucleotide labeled with a fluorescent dye such as the sulforhodamine101 (absorption maximum 594 nm, fluorescence 615 nm) by exciting lightderived from argon (Ar, 514 nm) or helium-neon (He—Ne, 545 nm).

[0099]FIGS. 17A through 17C are diagrams illustrating results of teststo verify the advantages of the substrate according to EMBODIMENT III.

[0100] Using a series of tested substrates, polynucleotides respectivelyhaving lengths of 521 bp and 625 bp, whose ends were labeled withfluorescent dye sulforhodamine 101 were hybridized to probes 311 and312, which probes had been respectively immobilized through silanecoupling layer 305 on each of the tested substrate. The treatedsubstrates were subjected to laser confocal microscopic scanning, andthe resultant relative fluorescence emission intensity and transmittanceare shown in FIG. 17A for a substrate according to EMBODIMENT IIIobtained by vacuum-depositing Cr 5.4 nm thick, FIG. 17B for a substrateobtained by vacuum-depositing Al 15 nm thick instead of Cr, and FIG. 17Cfor a substrate having no deposited metal film. In the figures, theabscesses indicate the scanning position on the substrate, and thevertical axes indicate, by solid line 330, fluorescence emissionintensity (equal to or more than 580 nm) distribution and transmittedlight intensity distribution detected with the laser microscope; and thebroken line 331 indicates transmittance distribution at wavelengths from380 nm to 450 nm.

[0101]FIG. 17A demonstrates that a constant fluorescence emissionintensity 330 could be obtained all over the area CA where thepolynucleotide labeled with the fluorescent dye sulforhodamine 101 washybridized, in the substrate according to EMBODIMENT III. Thefluorescence emission intensity 330 in the other positions wassufficiently low. The transmittance at 380 nm to 450 nm was almostconstant in any positions, indicating that the Cr layer was retainedwith stability and was not delaminated from the surface of glass 302over the reactions and procedures from immobilization of probes tohybridization of the target polynucleotide.

[0102]FIG. 17B for the Al-deposited substrate instead of Cr depositiondemonstrates that, the transmittance of the area CA where thepolynucleotide labeled with the fluorescent dye sulforhodamine 101 wasto be hybridized was remarkably high, indicating that the Al-depositedsurface had been dissolved in the immobilization of the probes. In otherwords, the Al-deposited surface was dissolved in a 10 mMTris-hydrochloric acid-1 mM EDTA buffer solution (pH 7.5) which is agenerally used medium for dissolving DNA and/or RNA, indicating thatsuch an Al-deposited substrate cannot be used in reaction systems usingaqueous solutions. Consequently, in this tested substrate, the probeswere not substantially immobilized, hence the polynucleotide labeledwith the fluorescent dye sulforhodamine 101 was not hybridized, and thefluorescence emission intensity was significantly low globally.

[0103] As is shown in FIG. 17C for the substrate where the probes wereimmobilized by subjecting the surface of the glass directly to silanecoupling, the area CA where the polynucleotide labeled with thefluorescent dye sulforhodamine 101 was hybridized had a higherfluorescence emission intensity 330 than the other positions where noprobes were immobilized, indicating that the substrate served toimmobilize the probes. The fluorescence emission intensity 330 in thissubstrate fluctuated, however, by location as compared with thesubstrate according to the present embodiment, indicating that theprobes were not immobilized homogeneously.

[0104] The above results demonstrates that the Cr-deposited substrateaccording to the present embodiment is advantageous for stableimmobilization of probes and for hybridization of targetpolynucleotides. In addition to Cr-deposited substrates, any substrateseach obtained by vacuum depositing a metal, which is stable in a weakalkali solution, on a glass substrate in a thickness of severalnanometers to 10 nm are advantageous. To be more specific, those coveredwith a film of oxide on the surfaces and hardly dissolved in a weakalkali solution at 95° C. are desirable, whereas gold and platinumcannot be used since they form no oxide film and hence silane couplingcannot be done. Stainless steel has similar characteristics to Cr suchas to be stable in a weak alkali solution and to form an oxide film, butit is excluded from the scope, since it does not allow active residuesto be introduced by silane coupling for some unknown reasons. Similarexaminations have revealed that at least any metal of Ti, V, Cr, Fe, Co,Ni, Mo and W is effective and advantageous.

[0105] Next, explanation will follow concerning that a targetpolynucleotide hybridized to a probe on the substrate according to thepresent embodiment can be efficiently extracted, with reference to FIGS.18A and 18B. FIGS. 18A and 18B respectively illustrate the results oftests where polynucleotides respectively having lengths of 521 bp and625 bp, whose ends were labeled with fluorescent dye sulforhodamine 101,were hybridized to probes 311 and 312, which probes were respectivelyimmobilized through silane coupling layer 305 on the areas 306 and 307shown in FIG. 16. The surface of each tested substrate was covered with20 microlitter of a 20 mM Tris-hydrochloric acid buffer (pH 7.5).

[0106]FIGS. 18A and 18B respectively demonstrate the results obtained bylaser scanning (514 nm) over the areas 306 and 307 separately anddetecting fluorescence emission intensity (510 nm to 580 nm), with theabscissa indicating scanning positions on the substrate and the verticalaxis indicating relative fluorescence emission intensity distributiondetected through a laser microscope. Line 335 represents the relativefluorescence emission intensity, indicating that a nearly constantfluorescence was detected all over the both areas 306 and 307. Next, thearea 306 alone was subjected to scanning with 10 mW YAG laser (spotdiameter 5 nm) at 1053 nm. As shown in FIG. 18A, the fluorescenceemission intensity after scanning was decreased to about one-tenth atthe region 333 which scanned with YAG laser, as indicated by broken line336. In the area 307, both fluorescence emission intensities 335 and 336did not change.

[0107]FIG. 19 schematically illustrates results of electrophoresisconcerning fractionation of polynucleotides according to the presentembodiment.

[0108] Lane 340 is a lane of a marker. Lanes 341 and 342 demonstrate theresults of electrophoresis on identical polynucleotides with thoseobtained in the areas 306 and 307 of the substrate 1 respectively. Bandsof 521 bp and 625 bp are clearly observed in the lanes 341 and 342,respectively. Lanes 343 and 344 demonstrate the results obtained byscanning the area 306 of the substrate 1 with 10-mW YAG laser (spotdiameter 5 nm) at 1053 nm, recovering the buffer on the surface of thescanned substrate 1, subjecting the buffer for PCR amplification, andsubjecting the PCR amplification products to 2% agarose gelelectrophoresis analysis. A band of 521 bp is clearly detected in thelane 343, whereas no band of 625 bp is detected in the lane 344,indicating that only the polynucleotide denatured from the area 306,which has been scanned with 10-mW YAG laser (spot diameter 5 nm), ispresent in a solution obtained by subjecting the recovered buffer to PCRamplification. According to the present embodiment, immobilization ofprobes and an efficient fractionation of hybridized polynucleotides canbe achieved.

[0109] Embodiment IV

[0110] EMBODIMENT IV is designed to utilize the inside wall of acapillary for minimizing the amount of a sample solution required forfractionation of polynucleotides, whereas the substrate 1 each in theabove EMBODIMENTS I through III is plane-shaped.

[0111]FIGS. 20 and 21 illustrate an external view and a sectional viewalong with the lines A-A of a capillary applicable in the fourthembodiment.

[0112] Capillary 401 is a light transmittable capillary such as a glasscapillary. Inside of capillary wall 412 is coated with inner coat 413composed of Cr or other metal exemplified in EMBODIMENT III. The surfaceof the inner coat is composed of a stable oxide formed by air oxidationof the metal. On the surface of the coat are cylindrically placed targetpolynucleotide hybridization areas 414, 415 . . . on which probes areimmobilized. Area specific probes are closely immobilized on each of thetarget polynucleotide hybridization areas 414, 415 . . . . Ofpolynucleotides in a sample solution, only each of polynucleotidescomplementary to each of the probes can be hybridized to each of theareas. On the external surface of the capillary 401 are indicatedmarkers 411 at the boundary between the individual areas so as to ensurethe observation of the positions of individual target polynucleotidehybridization areas from the outside of the capillary.

[0113]FIG. 22 is a diagram of an illustrative configuration forimmobilizing a probe on a target polynucleotide hybridization area.

[0114] Independent oligonucleotide probe layers can be formedconcentrically in the inner wall of a capillary, as illustrated in FIG.21, for example, in the following manner: Initially, epoxy groups 416are formed all over areas on the inner wall of the capillary 401 byreacting the whole inner wall of the capillary with a divalent reagenthaving silanol group and epoxy group at both ends to make the inner wallwater repellent and thereby reject an aqueous sample solution. Next, anucleotide sample solution to be hybridized to a specific area 415 isintroduced as a drop into the capillary 401. The drop becomes sphericaland in concentric contact with the inner wall with a minimum contactarea because of the water repellency of the inner wall of the capillary.The drop is then introduced to the area 415 and left for a while to makea free end of the epoxy group 416 to hybridize to an oligonucleotideprobe, which 5-end has been modified with an amino group. The dropcontaining the reacted nucleotide probe is taken out of the capillary401 immediately after the completion of the reaction. By repeatingprocedures in which a different sample drop is introduced to react inanother area in a similar manner, the configuration shown in FIG. 21 canbe obtained.

[0115] The timetable for fractionating target polynucleotides in asample solution according to the fourth embodiment is not describedherein as it is identical with the timetable shown in FIG. 15.

[0116]FIG. 23 is a general view of the configuration of a polynucleotideseparation apparatus according to the fourth embodiment. In the insideof polynucleotide separation module 431 is integrated the capillary 401described in FIGS. 20 and 21. Both ends of the capillary 401 areseparately connected to capillary connection units 432 and 433. One of asample solution retained in reservoir 434, a washing solution retainedin reservoir 435, a washing solution retained in reservoir 436 and airpassing through air filter 437 is selected by selector 438, andintroduced into the capillary connection unit 432 with pump 439.Controller 400 represents a control and power supply means according tothe apparatus of the present embodiment. The solution extracted from thepolynucleotide separation module 431 passes through the capillaryconnection unit 433 and is supplied to aftertreatment process 431including PCR amplification. To the controller 400 are collectednecessary data from individual units of the apparatus, and necessaryoperation signals in accordance with the data are sent to individualunits from the controller. Details of a practical configuration andconnection to individual units of the apparatus are omitted herein.These can be achieved by one skilled in the art based upon theaforementioned timetables and the following descriptions.

[0117]FIG. 24 is a sectional view along with the lines B-B of thepolynucleotide separation module 431 of the fourth embodiment. In theinside of the module 431 are provided, in parallel, light source 442 forintroducing a light to the wall of the capillary 401; doughnut-shapedheat sources 443 corresponding to individual target polynucleotidehybridization areas of the capillary 401; light detection probes 444 fordetecting fluorescence emitted from a target polynucleotidehybridization area which is excited by an exciting light plasmon-excitedfrom the capillary to the inside, or for detecting the markers 411 onthe surface of the capillary; and thermal detection probes 445 fordetecting the temperature of the capillary. Electrodes 451 and 452 areprovided respectively in the capillary connection units 432 and 433 toextract sample DNA in the capillary 401 by electrophoresis. Electrodeconnector 453 is to apply an electric potential to the inner coat 413 ofthe capillary 401 so as to move nucleotides toward or away from theinner surface of the capillary.

[0118]FIG. 25 illiterates a variation of the polynucleotide separationmodule 431 shown in FIG. 24 according to the fourth embodiment. Thebasic configuration of the variation is the same as in the embodimentshown in FIG. 24, except that the former employs drop 471 as a washingsolution to be introduced for extracting a separated polynucleotide. Thedrop 471 is introduced from the reservoir 436 into the capillary 401 byadjusting the selector 438 shown in FIG. 23; the drop 471 is then pushedto an objective target polynucleotide hybridization area by air passingthrough the air filter 437 so as to cover the area. Next, leak valves461 and 462 are opened to escape air in the capillary 401, which isexpanded by area specific heat from the heat source 443, the position ofthe drop 471 is thus fixed. The temperature of the area is thenincreased to a temperature equal to or higher than the denaturationtemperature to denature the target polynucleotide from the probe.Subsequently, the leak valves 461 and 462 are closed, and the drop 471is pushed to the capillary connection unit 433 by the air passingthrough the air filter 437 so as to extract the target polynucleotide.According to the present embodiment, the drop come in contact with aspecific target polynucleotide hybridization area alone, andcontamination to the drop will not occur even if the temperature ofother area reaches to the denaturation temperature. The extraction witha higher precision can, therefore, be achieved. In the presentembodiment, area specific heating is carried out by one of the heatsources 443 in the polynucleotide separation module 431, whereas theextraction with a high precision can also be achieved by using one heatsource in the shape covering all over the target polynucleotidehybridization areas.

[0119]FIG. 26 illustrates another variation of the polynucleotideseparation module 431 according to the embodiment shown in FIG. 24. Inthe present embodiment, light source 442, heat source 443, lightdetection probe 444 and heat detection probe 445 each to be provided inthe inside of the capillary 401 are omitted, and an optical system isprovided in the exterior to the capillary 401 as an alternative to theabove elements. In other words, the present embodiment employs thecapillary 401 instead of the substrate 1 in the configuration shown inFIGS. 2 and 6 according to EMBODIMENT I. Consequently, the identicaloptical system with that in FIG. 2 can be employed, whereas only theobjective lens 15 is shown in FIG. 26 for the simplification.

[0120] The capillary 401 connected to the capillary connection units 432and 433 is moved on the rail 446 to observe the state of the inside ofthe capillary at a specific target polynucleotide hybridization area;and simultaneously a convergent light is irradiated to theaforementioned area of the capillary to heat area-specifically thevicinity alone of the convergent point in the capillary. When thecapillary is designed to rotate in the circumferential direction, thestate of the hybridization of polynucleotide can be observed, heated andextracted all over the areas in the circumferential direction in thecapillary.

[0121]FIG. 27 illustrates a variation of the polynucleotide separationmodule 431 according to the embodiment shown in FIG. 26. The presentvariation is similar to the embodiment in FIG. 26 in that an opticalsystem is provided in the exterior of the capillary 401 but different inthat only one set of the optical system identical with those to beprovided in individual target polynucleotide hybridization areas in thecapillary is provided in the exterior of the capillary to therebyconstitute module 431, and that the module 431 is designed to be movedon rail 481 with respect to the capillary 401. By configuring like this,the apparatus can globally be miniaturized.

[0122] Embodiment V

[0123] The aforementioned EMBODIMENTS I through IV are applied to thecase where a target polynucleotide is contained in a sample solution,whereas EMBODIMENT V is directed to fractionate a target polynucleotidedirectly from cells themselves. According to the present embodiment,naturally, the target polynucleotide is finally fractionated in the formof a sample solution containing the same. Such a sample solution is,however, obtained after introducing cells to a target polynucleotidehybridization area so that the amount of target polynucleotide in thesample solution can be increased, and hence PCR or other pretreatmentcan be omitted in many cases. In addition, nucleotides, proteins andother cell components can be obtained for each of the cells according tothe present embodiment.

[0124]FIG. 28 illustrates basic elements of the present embodiment.

[0125] On the surface of substrate 1 is formed target polynucleotidehybridization area 511 as in described in the above EMBODIMENTS.Controller 521 is to apply a DC or alternating field between anelectrode of the hybridization area 511 and grounding electrode 514opposed to the substrate 1 to capture and hybridize cell 561 in thehybridization area 511. Power supply 526 is for applying a DC oralternating field or grounding, including selection switch 525. Peltierdevices 512 and temperature sensors 513 are embedded in the substrate 1separately in the vicinity of individual target polynucleotidehybridization areas. Temperature measurement and control unit 523 is toinput a signal from the temperature sensor 513 and to control thesurface temperatures of the individual target polynucleotidehybridization areas independently through Peltier devices 512.Controller 521 gives a direction on which area is to be controlled intemperature.

[0126]FIG. 29 illustrates a basic configuration according to the presentembodiment of a combination of a polynucleotide separation cell havingthe basic elements illustrated in FIG. 28 and an optical system. FIG. 30is a sectional view along with the lines A-A of the separation cellillustrated in FIG. 29.

[0127] Polynucleotide separation cell 541 is provided with sample inlettube 542 and sample outlet tube 543, through which sample cells, washingsolutions or the like are introduced into the separation cell, and cellresidues, proteins, purified nucleotide samples or the like arerecovered from the separation cell. In the inside of the separation cell541 are formed, as shown in FIG. 30, target polynucleotide hybridizationareas and are placed electrodes 551, 552, 553 . . . for introducing asample cell introduced from the sample inlet tube 542 to the targetpolynucleotide hybridization areas, and lines are connected to theelectrodes. In this figure, upper electrode 514 is not illustrated. Asingle stranded-oligonucleotide probe complementary to each targetpolynucleotide is individually immobilized in each target polynucleotidehybridization area.

[0128] The inside of the polynucleotide separation cell 541 can beobserved through the same optical system as described in FIG. 2. To bemore specific, the substrate 1 is placed in the direction perpendicularto the light axis of the objective lens 15, and an exciting light fromlight source 16 for fluorescent observation is induced via the bandpassfilter 151 and the dichroic mirror 181 to the objective lens 15. Theexciting light induced through the objective lens 15 excites afluorescent dye, which is attached accompanied with a targetpolynucleotide hybridized onto the substrate 1, and the excitedfluorescent dye emits fluorescence in an intensity in proportion to theamount of the target polynucleotide hybridized to probes on each area ofthe substrate. The emitted fluorescence in an intensity in proportion tothe amount of the target polynucleotide is collected through theobjective lens 15. Of the collected fluorescence, only a fluorescencehaving the wavelength to be detected is introduced via the emissionfilter 152 to the detector 191 to allow the observation of the inside ofthe separation cell 541.

[0129] In general, part of cell membrane of a cell is destroyed toliberate nucleotides, proteins and other components by heating.Consequently, by introducing a sample solution containing cells into thepolynucleotide separation cell 541 and thereafter increasing thetemperature of a specific target polynucleotide hybridization area onthe substrate 1 up to 95° C., a cell in the area is destroyed toliberate its components, and double stranded-polynucleotides can bedenatured into single stranded-polynucleotides. Subsequently, bydecreasing the temperature of the hybridization area to 37° C., a singlestranded-polynucleotide complementary to the probe in the area can behybridized to the probe, among these target polynucleotides. Thetemperature of a specific hybridization area can be controlled byselecting a Peltier device 512 corresponding to the area and passing anelectric current therethrough.

[0130] A process will be described for fractionating a cell componentand target polynucleotide directly from blood cells according to thepresent embodiment, with reference to FIGS. 31A through 31E and FIGS.32A through 32D.

[0131] Initially, the ion strength of a blood sample is decreased todestroy red blood cells, and thus a blood sample containing white bloodcells alone as cell components is prepared. FIG. 31A illustrates thestate where the target polynucleotide hybridization areas 551, 552, 553. . . are formed on the surface of the substrate 1. FIG. 31B illustratesthe state where the blood sample is introduced into the polynucleotideseparation cell 541 and white blood cells 561 float over the surface ofthe substrate 1. In this state, an alternating field is applied betweenthe electrodes of the target polynucleotide hybridization areas 551,552, 553 . . . and the electrode 514 so as to attract the white bloodcells 561 to the target polynucleotide hybridization areas. The gradientof electric field density generated between the electrode 514 and theelectrodes of individual hybridization areas is condensed at theindividual areas. An alternating field having a frequency of equal to ormore than 1 kHz and a density of equal to or more than 10 V/mm canpreferably used in this process. FIG. 31C illustrates the state wherewhite blood cells are induced to, and placed on the targetpolynucleotide hybridization areas. In this state, afluorescence-labeled antigen substance is introduced into the cell 541.FIG. 31D illustrates the state where only white blood cells capable ofmaking antibody response to the antigen substance (white blood cells 562and 563 hatched in the figure) are labeled and become to emitfluorescence. It is preferable in this state to cool the surface of thesubstrate to a temperature of about 4° C. and to ensure the amounts ofmRNA and the like in the cells not to change accompanied with thehybridization with the marker antigen. The hybridization capability ofthe antibodies on the surface of B cells or the like does not changewith a decreasing temperature. The position of a target polynucleotidehybridization area where a white blood cell emitting fluorescence can,therefore, be identified through the output of the detector 191. Thecomponent of a cell having reactivity with the antigen substance can beliberated into the sample solution by heating individual targetpolynucleotide hybridization areas in sequence to destroy the cells.FIG. 31E illustrates the state where only one white blood cell 563 ofthose hybridized on the individual target polynucleotide hybridizationareas is destroyed and disappears from the substrate 1. Accordingly, thecomponent of the specific white blood cell can be extracted byrecovering the sample solution in the polynucleotide separation cell 541while applying an alternating field to other cells in this state.

[0132]FIG. 32A illustrates the state where probes 671 are immobilized onone area 616 of the target polynucleotide hybridization areas. FIG. 32Billustrates the state where white blood cell 662 is induced andhybridized to the hybridization area 616 in the above manner. After thewhite blood cell 662 is induced to the hybridization area 616 as thus,the sample solution is exchanged with a new one to decrease its pH toabout 4. This is because almost all of pK values of proteins range 4 orhigher and by decreasing pH to 4 or lower, total charges of proteinsbecome positive whereas the charges of polynucleotides become negative;and hence the both can be separated in a DC field. Under such a pHcondition, the temperature of the hybridization area 616 is increased bythe Peltier device 512 to destroy the white blood cell. Thereafter, bysetting the electrode in the target polynucleotide hybridization area616 as an anode and the electrode 514 on the opposite surface as acathode, polynucleotides 664 are collected to the hybridization area 616and proteins 663 are electrophoresed to the face of the counterelectrode 514. FIG. 32C is a schematic diagram illustrating the statewhere polynucleotides 664 and proteins 663 migrate by electrophoresis inthe opposite direction to each other. The proteins can easily beseparated from polynucleotides by replacing the solution in the cell. Inthis process, polynucleotides complementary to the probe 671 immobilizedon the hybridization area naturally hybridize to the probes.

[0133] Next, by setting the electrode of the hybridization area as acathode and the counter electrode 514 as an anode, the targetpolynucleotide 666 hybridized to the probes 671 on the hybridizationarea 616 remains on the area 616, while polynucleotides not hybridizedare liberated into the sample solution. Of polynucleotides in the whiteblood cell 662, those not hybridized to the probes 671 can be extractedas in a sample solution by recovering the sample solution in the presentstep. FIG. 32D schematically illustrates the state where polynucleotides665 not hybridized to probes 671 migrate by electrophoresis.

[0134] Finally, a specific target polynucleotide hybridized to theprobes in a specific target polynucleotide hybridization area isliberated by increasing the temperature of the specific area up to about95° C. By recovering the sample solution in this step, the objectedtarget polynucleotide such as mRNA can be extracted and recovered.

[0135] After completion of the aforementioned procedure for on whiteblood cell, a next white cell is subjected to the same procedure toextract a mRNA or other target polynucleotide per each white blood cell.Separately, the extraction can be conducted by dying cells withdifferent plural types of markers by a marker labeling technique andobserving them with different filters to select and extract a cellmeeting a plurality of conditions.

[0136] The total amount of the target polynucleotide can quantitativelydetermined through signals from the optical system in the state wherethe target polynucleotide is hybridized to the probes in the targetpolynucleotide hybridization area. Consequently, the present embodimentcan be applied to the determination of the total amount of mRNA in a Bcell or other white blood cell, and according to the embodiment, acondition can be clarified easily and rapidly by estimating the activityof a white blood cell having reactivity to a specific antigen.

[0137] The present embodiment can naturally be applied to arbitrarycells in addition to white blood cells.

[0138] Other embodiments and variations will be obvious to those skilledin this art, this invention is not to be limited except as set forth inthe following claims.

1. A polynucleotide separation method comprising the steps of:immobilizing each of single stranded-oligonucleotide probes each havinga specific base sequence to each of a plurality of areas, said areasbeing independent and formed on the surface of a substrate, supplying asample solution containing polynucleotides onto said substrate, heatingsaid sample solution up to a predetermined temperature and thereaftercooling the heated solution to thereby hybridize each of complementarypolynucleotides separately to each of probes, replacing said samplesolution above the substrate with a solution containing nopolynucleotide, and heating the surface of the substrate at one area ofsaid plurality of independent areas on the substrate up to apredetermined temperature, and thereby denaturing only a polynucleotidebeing hybridized complementarily to said probe immobilized on said areato extract said denatured polynucleotide.
 2. A polynucleotide separationapparatus comprising: a substrate having a plurality of independentareas, each of single stranded-oligonucleotide probes each having aspecific base sequence being individually immobilized on each of saidareas, means for supplying a sample solution containing polynucleotidesonto said substrate, means for replacing said sample solution above thesubstrate with a solution containing no polynucleotide, temperaturecontrol means for heating said sample solution up to a predeterminedtemperature, temperature control means for heating [the sample solution]the surface of the substrate at only one area of said plurality ofindependent areas on the substrate to a predetermined temperature, andmeans for extracting said sample solution above the substrate.
 3. Apolynucleotide separation apparatus according to claim 2, furthercomprising means for quantitatively detecting, separately on each ofsaid areas, fluorescence emission intensity of a fluorescent dye withrespect to each of said polynucleotides hybridized to said each probe ofareas on the substrate, or intensity of autoemission fluorescence ofsaid polynucleotides.
 4. A polynucleotide separation apparatus accordingto claim 3, wherein a light having wavelengths ranging from 280 nm to650 nm is used as an exciting light for said observation offluorescence.
 5. A polynucleotide separation apparatus according toclaim 3, further comprising means for analyzing the temperatures of eachof said areas separately based on changes of said quantitativelydetected fluorescence emission intensity of the fluorescent dye attachedto said individual polynucleotides hybridized separately to each of saidareas of the substrate or to said surface of the substrate, or based onchanges of said quantitatively detected fluorescence autoemissionintensity of said nucleotide sample.
 6. A polynucleotide separationapparatus according to claim 5, further comprising means forfeedback-controlling the temperature of a specific area on the surfaceof the substrate separately, based on the analysis result concerning thetemperature of each of said areas of the substrate obtained through saidmeans for detecting fluorescence emission intensity emitted from thefluorescent dye.
 7. A polynucleotide separation apparatus according toclaim 2, further comprising means for separately analyzing saidtemperature of each area of the substrate through a thermistor or athermocouple.
 8. A polynucleotide separation apparatus according toclaim 7, further comprising means for separately feedback-controllingthe temperature of a specific area on the surface of the substrate,based upon said analysis result on the temperature of each of said areasof the substrate obtained through said analyzing means.
 9. Apolynucleotide separation apparatus according to claim 2, furthercomprising thin film layers or particle layers having highphotoabsorbing characteristics, each layer being formed separately ateach of said areas on the substrate, and means for selectivelyirradiating a convergent light to a thin film layer or particle layer atsaid specific area of the substrate, wherein said specific microarea isarea-specifically heated through the photoabsorption of the light beingselectively irradiated.
 10. A polynucleotide separation apparatusaccording to claim 9, wherein a light having a wavelength being notabsorbed by any nucleotides is used as said convergent light for heatingsaid microarea.
 11. A polynucleotide separation apparatus according toclaim 9, wherein a substance absorbing lights each having a wavelengthlonger than 400 nm is applied, sprayed or vacuum-deposited on asubstrate having a plurality of independent areas on its surface, andeach of single stranded-oligonucleotide probes each having a specificbase sequence being individually immobilized to each of said areas. 12.A polynucleotide separation apparatus according to claim 9, wherein saidexciting light used for the excitation in the florescent observation,said light for fluorescent observation and said convergent light forheating the microarea individually have a different wavelength from eachother.
 13. A polynucleotide separation apparatus according to claim 2,further comprising a microsphere having an extremely higherphotoabsorbing characteristics than the substrate, means for capturingsaid microsphere floating in the solution through a light radiationpressure generated by a convergent light having a numerical aperture ofequal to or more than 1.2, and means for moving arbitrarily saidmicrosphere to the vicinity of said specific area on the substrate bysaid capturing means through the light radiation pressure and forheating said specific area on the substrate area-specifically.
 14. Apolynucleotide separation apparatus according to claim 2, furthercomprising an array of heating element layers, each layer being attachedto each of said areas of the substrate, and means for area-specificallyheating said specific microarea by allowing one of said heating elementlayers to evolve heat.
 15. A polynucleotide separation apparatusaccording to claim 2, wherein said substrate is in the form of capillaryhaving, on its inner surface, a plurality of independent split areas,each of different nucleotide probes being immobilized on each of saidareas, and which apparatus comprising means for introducing a samplenucleotide solution into said capillary, temperature control means forhybridizing said probes to polynucleotide components in said samplesolution, means for removing polynucleotides in the sample solution,which polynucleotides being not hybridized to said probes on the surfaceof the capillary, means for heating a specific area of said plurality ofareas in the capillary to denature said polynucleotide component in thesample solution, said component having been hybridized to the probe atsaid area, and means for extracting said denatured polynucleotidecomponent.
 16. A polynucleotide separation apparatus according to claim15, comprising said means for introducing a sample nucleotide solutioninto said capillary, said temperature control means for hybridizing saidprobes to polynucleotide components in said sample solution, said meansfor removing polynucleotides in the sample solution, saidpolynucleotides being not hybridized to said probes on the surface ofthe capillary, means for placing a drop containing no nucleotide to comein contact with only a specific area of said plurality of areas in thecapillary, means for heating said capillary to denature only said samplesolution component being hybridized to the nucleotide probe at the area,said drop being in contact with said area, and means for extracting thedrop containing said denatured nucleotide component.
 17. Apolynucleotide separation apparatus according to claim 16, comprisingsaid capillary having, on its inner surface, a plurality ofcylindrically split independent areas, each of different nucleotideprobes being immobilized to each of said areas, means for introducing asample solution, a washing solution and air to said capillary, means forindividually heating each of said areas of the capillary, means forheating said specific area of the capillary and denaturing said samplesolution component being hybridized to the nucleotide probe at said areato extract said component.
 18. A polynucleotide separation apparatusaccording to claim 2, wherein said substrate has a metal thin film layeron its surface, and said probes are separately immobilized through ametal oxide formed on the surface of said metal thin film layer and acrosslinking agent.
 19. A polynucleotide separation apparatus accordingto claim 18, wherein said metal oxide film layer absorbs a coherentlight or a light having continuous wavelength, each having a wavelengthof equal to or more than 350 nm and less than 633 nm.
 20. Apolynucleotide separation apparatus according to claim 18, wherein saidmetal oxide film layer absorbs a coherent light or a light havingcontinuous wavelength each having a wavelength of equal to or more than633 nm and equal to or less than 1053 nm.
 21. A polynucleotideseparation apparatus according to claim 18, further comprising a metalsurface composed of an active residue A, a linker R and a metal Mehaving an oxidized surface and of the formula A—R—O—Me, wherein saidpolynucleotide probes are immobilized through said active residue A. 22.A polynucleotide separation apparatus according to claim 21, wherein theactive residue is introduced onto the oxide surface of the metal througha silane coupling reagent, and said polynucleotide probes areimmobilized individually through said active residue.
 23. Apolynucleotide separation apparatus according to claim 21, wherein saidactive residue A is a glycidoxy group, and wherein polynucleotide probeseach having an amino group are immobilized individually through saidactive residue A.
 24. A polynucleotide separation apparatus according toclaim 18, wherein said metal oxide film layer has any oxide of a metalselected from the group consisting of Cr, Ti, V, Fe, Co, Ni, Mo and W.25. A polynucleotide separation apparatus according to claim 2, furthercomprising means for applying a DC field onto the surface of saidsubstrate.
 26. A polynucleotide separation apparatus according to claim25, further comprising means for applying said DC field while allowingthe pH of the solution containing the sample to equal to or lower than 4to attract nucleotide components alone to the surface of the substratemodified with nucleotide probes.
 27. A polynucleotide separationapparatus according to claim 2, further comprising means for applying analternating field onto the surface of said substrate.
 28. Apolynucleotide separation apparatus according to claim 2, furthercomprising a reservoir for retaining the sample solution, a substratehaving a plurality of two-dimensionally split areas on its surface, eachof said areas being modified with an oligonucleotide probe, means forapplying an alternating or DC field individually to each of said areasof the substrate, means for allowing individually each of said areas ofthe substrate to evolve heat, and means for identifying an area wheresaid hybridized cell being present and for verifying the position of acell dyed with a marker.
 29. A polynucleotide separation apparatusaccording to claim 2, wherein heating means capable of individuallyheating each of said areas to a temperature ranging from 60° C. to 95°C. is used.